A Sensitive and Automated Diagnostic Assay for Lung Cancer Lung cancer is the number one cancer killer in the USA. The ability to identify lung cancer at its early, curable stages will reduce the mortality and is thus an important clinical goal. Toward this goal, we have identified a panel of genetic biomarkers that can be detected in sputum for early detection of lung cancer (AACR Breaking News). We have also developed an in situ mini-chip comprising multiple fluorescein-labeled probes that can measure the genetic biomarkers in a single test. However, the fluorescent signals of the probes are weak and fade fast, reducing the sensitivity of the biomarkers. Furthermore, manually counting the blurry signals of the multiple probes is time- consuming and labor intensive with high variability, thus limiting the promising diagnostic approach in clinical setting. The recent studies have shown that fluorophore-metal interaction, namely, metal-enhanced fluorescence (MEF), can increase intensity and photostability of fluorescent signals, and an emerging automated dot counting system permits rapidly and objectively analyze multiple probes. The objective of the R21 exploratory project is to develop the in situ mini-chip as a robust diagnostic assay that can reliably detect the genetic biomarkers for the early detection of lung cancer in sputum samples. We propose to 1) increase the sensitivity of the in situ mini-chip by hybridizing the fluorescent probes to the cells immobilized on the slides coated with silver nanoparticles, 2) develop the in situ mini-chip as robust assay by analyzing the enhanced fluorescent signals in sputum cells using the automated dot-counting system, and 3) determine diagnostic performance of the robust assay for early detection of lung cancer using sputum samples in our existing case-control cohort of patients with stage I lung cancer. At completion, the in situ mini-chip will be refined as a reliable, automated, and noninvasive genetic test for lung cancer early detection. The study will lay the basis for large prospective clinical project (R01) or multicenter cooperative clinical study (R33) designed to further evaluate its full utility that could rapidly and widely be accepted and applied in routine clinical laboratories. This exploratory study will bring together complementary strengths of scientists and clinician with extensive expertise in molecular genetics, nanotechnology, cytopathology, oncology, and biostatistics. [unreadable] [unreadable] [unreadable]